Starch is the major form of carbon reserve in plants, constituting 50% or more of the dry weight of many storage organs—e. g. tubers, seeds of cereals. Starch is used in numerous food and industrial applications. In many cases, however, it is necessary to modify the native starches, via chemical or physical means, in order to produce distinct properties to suit particular applications. It would be highly desirable to be able to produce starches with the required properties directly in the plant, thereby removing the need for additional modification. To achieve this via genetic engineering requires knowledge of the metabolic pathway of starch biosynthesis. This includes characterisation of genes and encoded gene products which catalyse the synthesis of starch. Knowledge about the regulation of starch biosynthesis raises the possibility of “re-programming” biosynthetic pathways to create starches with novel properties that could have new commercial applications.
The commercially useful properties of starch derive from the ability of the native granular form to swell and absorb water upon suitable treatment. Usually heat is required to cause granules to swell in a process known as gelatinization, which has been defined (W A Atwell et al, Cereal Foods World 33, 306-311, 1988) as “ . . . the collapse (disruption) of molecular orders within the starch granule manifested in irreversible changes in properties such as granular swelling, native crystallite melting, loss of birefringence, and starch solubilization. The point of initial gelatinization and the range over which it occurs is governed by starch concentration, method of observation, granule type, and heterogeneities within the granule population under observation”. A number of techniques are available for the determination of gelatinization as induced by heating, a convenient and accurate method being differential scanning calorimetry, which detects the temperature range and enthalpy associated with the collapse of molecular orders within the granule. To obtain accurate and meaningful results, the peak and/or onset temperature of the endotherm observed by differential scanning calorimetry is usually determined.
The consequence of the collapse of molecular orders within starch granules is that the granules are capable of taking up water in a process known as pasting, which has been defined (W A Atwell et al, Cereal Foods World 33, 306-311, 1988) as “ . . . the phenomenon following gelatinization in the dissolution of starch. It involves granular swelling, exudation of molecular components from the granule, and eventually, total disruption of the granules”. The best method of evaluating pasting properties is considered to be the viscoamylograph (Atwell et al, 1988 cited above) in which the viscosity of a stirred starch suspension is monitored under a defined time/temperature regime. A typical viscoamylograph profile for potato starch shows an initial rise in viscosity, which is considered to be due to granule swelling. In addition to the overall shape of the viscosity response in a viscoamylograph, a convenient quantitative measure is the temperature of initial viscosity development (onset). FIG. 1 shows such a typical viscosity profile for potato starch, during and after cooking, and includes stages A-D which correspond to viscosity onset (A), maximum viscosity (B), complete dispersion (C) and reassociation of molecules (or retrogradation, D). In the figure, the dotted line represents viscosity (in stirring number units) of a 10% w/w starch suspension and the unbroken line shows the temperature in degrees centigrade. At a certain point, defined by the viscosity peak, granule swelling is so extensive that the resulting highly expanded structures are susceptible to mechanically-induced fragmentation under the stirring conditions used. With increased heating and holding at 95° C., further reduction in viscosity is observed due to increased fragmentation of swollen granules. This general profile has previously always been found for native potato starch.
After heating starches in water to 95° C. and holding at that temperature (for typically 15 minutes), subsequent cooling to 50° C. results in an increase in viscosity due to the process of retrogradation or set-back. Retrogradation (or set-back) is defined (Atwell et al., 1988 cited above) as “ . . . a process which occurs when the molecules comprising gelatinised starch begin to reassociate in an ordered structure . . . ”. At 50° C., it is primarily the amylose component which reassociates, as indicated by the increase in viscoamylograph viscosity for starch from normal maize (21.6% amylose) compared with starch from waxy maize (1.1% amylose) as shown in FIG. 2. FIG. 2 is a viscoamylograph of 10% w/w starch suspensions from waxy maize (solid line), conventional maize (dots and dashes), high amylose variety (HYLON® V starch, dotted line) and a very high amylose variety (HYLON® VII starch, crosses). The temperature profile is also shown by a solid line, as in FIG. 1. The extent of viscosity increase in the viscoamylograph on cooling and holding at 50° C. depends on the amount of amylose which is able to reassociate due to its exudation from starch granules during the gelatinization and pasting processes. A characteristic of amylose-rich starches from maize plants is that very little amylose is exuded from granules by gelatinization and pasting up to 95° C., probably due to the restricted swelling of the granules. This is illustrated in FIG. 2 which shows low viscosities for a high amylose (44.9%) starch (HYLON® V starch) from maize during gelatinization and pasting at 95° C. and little increase in viscosity on cooling and holding at 50° C. This effect is more extreme for a higher amylose content (58%, as in HYLON® VII starch), which shows even lower viscosities in the viscoamylograph test (FIG. 2). For commercially-available high amylose starches (currently available from maize plants, such as those described above), processing at greater than 100° C. is usually necessary in order to generate the benefits of high amylose contents with respect to increased rates and strengths of reassociation, but use of such high temperatures is energetically unfavourable and costly. Accordingly, there is an unmet need for starches of high amylose content which can be processed below 100° C. and still show enhanced levels of reassociation, as indicated for example by viscoamylograph measurements.
The properties of potato starch are useful in a variety of both food and non-food (paper, textiles, adhesives etc.) applications. However, for many applications, properties are not optimum and various chemical and physical modifications well known in the art are undertaken in order to improve useful properties. Two types of property manipulation which would be of use are: the controlled alteration of gelatinization and pasting temperatures; and starches which suffer less granular fragmentation during pasting than conventional starches.
Currently the only ways of manipulating the gelatinization and pasting temperatures of potato starch are by the inclusion of additives such as sugars, polyhydroxy compounds of salts (Evans & Haisman, Starke 34, 224-231, 1982) or by extensive physical or chemical pre-treatments (e.g. Stute, Starke 44, 205-214, 1992). The reduction of granule fragmentation during pasting can be achieved either by extensive physical pretreatments (Stute, Starke 44, 205-214, 1992) or by chemical cross-linking. Such processes are inconvenient and inefficient. It is therefore desirable to obtain plants which produce starch which intrinsically possesses such advantageous properties.
Starch consists of two main polysaccharides, amylose and amylopectin. Amylose is a generally linear polymer containing α-1,4 linked glucose units, while amylopectin is a highly branched polymer consisting of a α-1,4 linked glucan backbone with α-1,6 linked glucan branches. In most plant storage reserves amylopectin constitutes about 75% of the starch content. Amylopectin is synthesized by the concerted action of soluble starch synthase and starch branching enzyme [α-1,4 glucan: α-1,4 glucan 6-glycosyltransferase, EC 2.4.1.18]. Starch branching enzyme (SBE) hydrolyses α-1,4 linkages and rejoins the cleaved glucan, via an α-1,6 linkage, to an acceptor chain to produce a branched structure. The physical properties of starch are strongly affected by the relative abundance of amylose and amylopectin, and SBE is therefore a crucial enzyme in determining both the quantity and quality of starches produced in plant systems.
In most plants studied to date e.g. maize (Boyer & Preiss, 1978 Biochem. Biophys. Res. Comm. 80, 169-175), rice (Smyth, 1988 Plant Sci. 57, 1-8) and pea (Smith, Planta 175, 270-279), two forms of SBE have been identified, each encoded by a separate gene. A recent review by Burton et al., (1995 The Plant Journal 7, 3-15) has demonstrated that the two forms of SBE constitute distinct classes of the enzyme such that, in general, enzymes of the same class from different plants may exhibit greater similarity than enzymes of different classes from the same plant. In their review, Burton et al. termed the two respective enzyme families class “A” and class “B”, and the reader is referred thereto (and to the references cited therein) for a detailed discussion of the distinctions between the two classes. One general distinction of note would appear to be the presence, in class A SBE molecules, of a flexible N-terminal domain, which is not found in class B molecules. The distinctions noted by Burton et al. are relied on herein to define class A and class B SBE molecules, which terms are to be interpreted accordingly.
However in potato, only one isoform of the SBE molecule (belonging to class B) has thus far been reported and only one gene cloned (Blennow & Johansson, 1991 Phytochem. 30, 437-444, and Koβmann et al., 1991 Mol. Gen. Genet. 230, 39-44). Further, published attempts to modify the properties of starch in potato plants (by preventing expression of the single known SBE) have generally not succeeded (e.g. Müller-Rober & Koβmann 1994 Plant Cell and Environment 17, 601-613).